Wolbachia are cytoplasmically inherited bacteria that have spread through thousands of animal species by a host manipulation called cytoplasmic incompatibility (CI). CI is a postmating incompatibility that renders Wolbachia-infected males incompatible with uninfected females. However, matings between infected males and infected females are compatible, thereby imparting a fitness advantage to infected females. The molecular mechanism that underpins CI has evaded biologists for almost two decades, despite obvious, applications to the evolution of host-bacterial interactions, genetic conflict, animal speciation, and vector control strategies to curb human diseases. The goal of this project is to investigate a new hypothesis for the molecular mechanism of CI. We propose that CI results from an infectious epigenetic alteration in which Wolbachia encrypt the sperm and egg through a manipulation of DNA methylation. This hypothesis is based on three key points: (i) First, since Wolbachia are stripped from the sperm in the cytoplasmic waste bag during spermatogenesis, CI must involve a modification of the sperm in the testes of a Wolbachia-infected male. The eggs of an infected female must also be able to rescue the Wolbachia-sperm modification. These gender-specific encryptions are hallmarks of epigenetic phenomena. (ii) Second, the embryonic inviability that results from a CI cross occurs during and preceding the first mitiotic division of the fertilized egg, where epigenetic phenomena are known to be important. (iii) Third, our preliminary evidence shows that infected Drosophila melanogaster flies treated with a small molecule inhibitor of DNA methylation (5-Azacytidine) exhibit CI when only one gender is treated with the drug. This effect, however, is not observed in uninfected, control crosses. Furthermore, treatment of both infected males and females leads to a restoration of compatibility, suggesting that differences in DNA methylation levels between the maternal and paternal genome lead to CI. To determine if Wolbachia alters DNA methylation to cause CI, we will bisulfite sequence the entire genome at 80X coverage to compare DNA methylation patterns in Wolbachia-infected and uninfected flies. Since DNA methylation and gene expression are tightly regulated, we will next sequence the whole transcriptome with RNA-seq from the same samples to identify differences in gene expression between Wolbachia-infected and uninfected flies that can be correlated back to the differences in DNA methylation based on infection status. Finally, we will determine if conserved, animal DNA methylation and demethylation genes are required for the modification by Wolbachia. This research will determine, for the first time, if Wolbachia induces an infectious epigenetic alteration to cause CI.